Engineered tissues, especially human tissues, offer great hope for treatment of a variety of diseases and for repair of damage to natural tissues due to trauma. Adults produce about 400 billion cycling cells daily, and loss of production of these cells is life threatening. In treating cancer patients, chemotherapy and radiation are commonly used; however, high-dose toxic drugs and irradiation not only kill cancer cells but also healthy hematopoietic cells produced by human bone marrow. Red blood cell and platelet transfusion and hematopoietic stem cell and progenitor cell (HSC/HPC) infusion are two commonly used clinical methods to replace mature blood cells and to reconstitute the blood-producing capacity of the patient. However, allogeneic matched donors are difficult to find and autologous HSC/HPC may be contraindicated or limited. Cord blood (CB) cells are obtained from umbilical cord of newborn babies and are rich in CD34+ cells and easy to procure. The challenge is to expand CB cells to large enough quantities for adult patients or for repeated transplant. Attempted improvements to ex vivo expansion methodologies have included the use of exogenous cytokines or growth factors or altering culture parameters such as culture duration and feeding schedules, which influence the differentiation and self-renewal of HSC/HPC. The ideas of these studies were to mimic the key elements of in vivo hematopoiesis environment, including soluble factors (e.g., cytokines), support cells and adhesion molecules, and physiochemical parameters.
However, to date, the field of hematotherapy has not extensively examined the effects of three-dimensional (3-D) geometry of the in vivo HSC/HPC environment and has failed to replicate the 3-D geometry in the ex vivo expansion systems. It is well established that cellular activities, including migration, proliferation, differentiation, and tissue functions, are significantly affected by cellular organization and structural cues both in vivo and in ex vivo cultures. Based on this knowledge, we have developed a 3-D small-scale culture system for ex vivo growth of HSC/HPC using a polyethylene terephthalate (PET) non-woven matrix. Our preliminary data show a substantial advantage for growth of CD34+ positive cells and committed colony forming units (CFU) in this 3-D matrix compared to standard two-dimensional control cultures. To successfully apply the 3-D culture system for clinical use, a perfusion bioreactor system is critically needed. The perfusion bioreactor system provides an environment for continuous nutrient delivery and waste removal, and thus sustains a high cell density in a 3-D matrix over an extended culture period. In addition, the perfusion bioreactor system can be automatically controlled and is less demanding for operation and culture handling, an important requirement for clinical use.
Also of interest are the cells in human bone marrow, as bone marrow contains hematopoietic tissue and the associated supporting stroma. While the hematopoietic stem cells produce mature blood cells, marrow stromal or stem cells (MSC) are the progenitor cells of skeletal tissue components and have the ability to differentiate into cell types phenotypically unrelated such as osteocytes, chondrocytes, muscle cells, adipocytes, and cardiomyocytes. Propelled by an increasing knowledge of human mesenchymal stromal cells (hMSC) biology, clinical evidence is emerging in the literature suggesting the tantalizing potential of hMSC for treating a wide range of diseases including osteogenesis imperfecta, tendon repair, stroke, and heart failure (8-11). For example, hMSC can be converted into myogenic progenitors in response to physiological stimuli, thus providing an alternative strategy for treatment of muscle dystrophies (3). In a recent study, researchers showed that injected bone marrow cells can form myocardial tissue and partially restored lost heart function in mice (24). To utilize hMSCs in clinical practice, a major obstacle, however, is to expand them to large quantity and yet retain their differentiation potential during the expansion.
The multi-potential properties of hMSC were first observed in the mid-1970s when whole bone marrow was grown in plastic culture dishes (18; 19). A small fraction of cells can be easily isolated by their adherence to the plastic surface after non-adherent blood cells were poured off. These adherent cells exhibited heterogeneous appearance and possessed striking features of self-renewal and differentiation even after 20 to 30 cell doublings (20). Recently, a more homogeneous (98% at passage 2) population of human MSC was obtained from bone marrow by using a density gradient to eliminate unwanted cell types (2). The cell population was expanded extensively on plastic culture dish and it maintained the ability to differentiate into multiple cell types in vitro, including adipogenic, chondrogenic, and osteogenic lineages (2). In a detailed study on purified hMSC growth kinetics, selfrenewal, and the osteogenic differentiation, hMSC was expanded for over 1.2×109 folds for 10 passages and maintained osteogenic differentiation capacity (4). However, a gradual increasing replicative senescence determined by the loss of population doubling potential after the first passage was observed. A recent study also reported a diminishing proliferation rate and a gradual loss of hMSC's differentiation capacity. The average doubling time increased from 1.3 for fresh bone marrow to 7.7 at passage 1 and up to 15.8 at passage 5, whereas adipogenesis started fading by 18 doublings and is totally lost by 22 passages (13). Studies have found that seeding density has profound effects on the growth rate of plastic-adherent cells from human bone marrow (12; 23). When the purified hMSCs were plated at low densities of 1.5 to 3.0 cells/cm2, they generated single-cell derived colonies and amplified about 109-fold in 6 wk (12). These single-cell-derived colonies contained three morphologically distinct cell types: spindle-shaped cells, large flat cells, and very small round cells, suggesting a heterogeneous cell population (12; 23). Compared to large cells, small round cells have greater rate of replication and enhanced potential for multi-lineage differentiation (23). The heterogeneous cell population and sensitivity to plating density were also observed for rat marrow stromal cells (14). Cell growth sensitivity to plating density may be explained by cell—cell contact and low seeding density appears to greatly enhance hMSC self-renewal and retain differentiation potential.
However, prior protocols used in obtaining and expanding hMSC require frequent cell passages and very large surface area for cells to grow. They are not designed for ex vivo expanding a large quantity of hMSC for clinical use. The 3-D feature, which is characteristic for hMSC in vivo environment, is also missing in the preparation. Many clinical cases require the reconstruction of a functional tissue in vitro before being transplanted to replace the damaged one. This becomes especially critical when the defect is larger than those that would spontaneously heal such as a large area of skin and a large bone defect, or when the immediumte replacement of tissue function is needed such as the replacement of cardiac muscle function. In these cases, a large number of cells alone are not sufficient; the cell must also exhibit desired functions or can be induced into a functional state once placed in the injury site. To achieve this, 3-D culture systems offer many advantages over conventional 2-D culture systems. A 3-D matrix offers a high surface area per unit volume and captures the 3-D feature of the in vivo tissue. Matrices that offer a 3-D structure have been widely used for tissue engineering a wide variety of tissues and for ex vivo expanding human hematopoietic progenitors (25-36). Among the materials used in these studies, non-woven fibrous matrix offers unique advantages. The non-woven fibrous matrix has isotropic structure, e.g., it has the same properties at three coordinates. In these matrices, regardless of where a cell lands, there will be the same amount of surface area available to it and it would have the same opportunity to interact with other cells. It also provides an environment where cells will have intimate interactions with neighboring cells and ECM network, which is a defining feature of in vivo tissue.
Three-dimensional matrices such as collagen gels, porous gelatin sponges, porous hydroxyapatite ceramic carrier, and a composite of hydroxyaptite/tricalcium phosphate (HA/TCP) particles have been previously investigated for cartilage and bone regeneration from hMSC (37-40). Particle size and shape, seeding density, and contraction kinetics influenced the growth and secretion of ECM proteins by hMSC. In these studies, hMSCs were first expanded in culture and then loaded onto the matrices. The end results were evaluated based on the performance after implantation. Despite the successes, a number of questions remain to be answered. The ex vivo expansion of hMSC is not carried out on these matrices. It also lacks the detailed information on how hMSCs adhere to the surfaces of the scaffolds and how the structures of the scaffolds affect their proliferation. A single device combining hMSC isolation, adhesion, expansion, and modularity will be of great advantage in simplifying the operation, especially in clinical use. In addition, hMSC grown at high density in a 3-D matrix may be directly induced to differentiate into desired functional tissues and be used in repairing large wounds. The critical requirements for the 3-D expansion system are high yield for hMSC isolation, high expansion rate, maintenance of primitiveness, and formation of desired tissue structure. For clinical use, the system should meet these requirements in one single unit.